ZIA BC 010600 (ZIA) | |||
---|---|---|---|
Title | AAA Proteins, Their Functions and Related Diseases | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Xia, Di | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $215,333 | Project Dates | null - null |
Fiscal Year | 2018 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Aging (100.0%) Cancer (100.0%) |
Brain (100.0%) Nervous System (100.0%) |
||
Research Type | |||
Normal Functioning Resources & Infrastructure Related to Biology |
|||
Abstract | |||
Our recent work has been focusing on the human AAA protein p97, the major cytosolic AAA chaperone. Although it has been known that D2 ring of p97 contributes most to the overall ATPase activity of p97, the function of the D1 ring is not clear. Our work has contributed significantly to function of the D1 ring, which is the regulatory domain of the p97. This result came from our study of p97 mutants that cause IBMPFD or MSP1. IBMPFD mutants have single amino acid substitutions at the interface between the N-terminal domain (N-domain) and the adjacent AAA domain (D1) and our work suggests that the mutations result in a reduced affinity for ADP. The structures of p97 N-D1 fragments bearing IBMPFD mutations adopt an Up N-domain conformation or Up-conformation in the presence of Mg2+-ATPgS, which is reversible by ADP (Down-conformation), demonstrating for the first time the nucleotide-dependent conformational change of the N-domain. The transition from the ADP- to the ATPgS-bound state is accompanied by a loop-to-helix conversion in the N-D1 linker and by an apparent re-ordering in the N-terminal region of p97. X-ray scattering experiments suggest that wild type p97 subunits undergo a similar nucleotide-dependent N-domain conformational change. We propose that IBMPFD mutations, by destabilizing the ADP bound form, alter the timing of the transition between nucleotide states and consequently interfere with the interactions between the N-domains and their substrates. Wild type and mutant N-D1 fragments were also studied in the presence of ATPgS or ADP in solution by SAXS, suggesting that nucleotide-dependent Up- and Down-N-domain conformational change also takes place in solution. Using isothermal titration calorimetry (ITC), we determined a Kd value of 0.88 uM towards ADP for the wild type N-D1 with a stoichiometry of 0.35, suggesting only 2 out of 6 sites are available for binding, which is consistent with previously reports of occluded ADP in wild-type p97. By contrast, mutant p97 N-D1 fragments displayed reduced binding affinities for ADP. For example, the R155H mutant showed a maximum reduction with a Kd of 4.25 uM. Notably, the amount of occluded ADP in mutant p97 is dramatically reduced. Unexpectedly, the titration profiles with ATPgS for mutants were biphasic and can only be fitted to a two-site model. The Kd values for the high affinity site were well determined and close to 0.1 uM for all mutants, whereas those for the low affinity site were associated with significant errors. Again, mutant p97 displayed higher stoichiometry than wild type in the ATPgS titration experiments. A model with four nucleotide-binding states for the ATP cycle in the D1-domain was proposed. First, there is an ATP state, with ATP bound and the N-domain in the Up-conformation. In a wild type p97 hexamer, due to non-exchangeable, pre-bound ADP, not all subunits will have their N-domains in the Up-conformation even with an excess amount of ATP in solution. We therefore hypothesize that there is an ADP-locked state, with non-exchangeable, pre-bound ADP at the D1 site and the N-domain in the Down-conformation. This state appears to be important for wild type p97 function and the pre-bound ADP is particularly difficult to exchange. The structure of the N-D1 fragment of wild type p97 may represent this conformation. In a third state, termed ADP-open, ADP is bound but exchangeable. This state was observed for mutant p97 by its biphasic ITC titration profile and is presumably in equilibration with the ADP-locked state. The structure of R155H with bound ADP represents this conformation. The fourth state is the Empty state, with nucleotide-binding sites unoccupied and the N-domain in an unknown position. The difference between the wild type and mutants, however, lies in the transition between the ADP-locked state and the ADP-open state. We propose that in the wild type protein this transition is tightly controlled and characterized by the asymmetry in nucleotid |